Histochemical studies of uric acid in some insects. 2. Uric acid and polyphenols in the fat body

It is recalled that in the larva of Aedes aegypti, starved after a rich protein diet, uric acid is formed and accumulates in the fat body, not as solid spheres but in high concentration in aqueous vacuoles. In the mature larva of Celliphora vicina which has finished feeding and is settling down to form the puparium, the fat body at first contains no argentaffin deposits. During the following 2 or 3 days, argentaffin material appears in the form of amber or brown vesicles and black granules of all sizes. Some of this material remains in the fat body cells; but a large part, presumably polyphenols, is discharged from the cells so that finally all the amber staining disappears and only black granules remain. During this transfer the epidermal cells become charged with sclerotin precursors, which are transferred into the outer part of the cuticle to form the puparium. The stored uric acid remains in the fat body and is dispersed during adult development and ultimately excreted.     

A new aspect of view in synthesizing new type beta-adrenoceptor blockers with ancillary antioxidant activities.

A series of vanilloid-type beta-adrenoceptor blockers derived from antioxidant traditional Chinese herbal medicines were synthesized and tested for their antioxidant and adrenoceptor antagonistic activities. They all possessed significant beta-adrenoceptor blocking activities under in vitro experiments and radioligand binding assays. In addition, some compounds were further examined in in vivo tests and produced antagonist effects matching that of propranolol and labetalol by measurements of antagonism toward (-)isoproterenol-induced tachycardia and (-)phenylephrine-induced pressor responses in anesthetized rats. Furthermore, all of the compounds had antioxidant effects inherited from their original structures. In conclusion, compound 11 had the most potent beta-adrenoceptors blocking activity, 12 and 13 possessed high cardioselectivity, whereas 14, 15 and 16 possessed additional alpha-adrenoceptor blocking activity and 15 is the most effective antioxidant of all. The antioxidant activity may be due to their alpha and beta unsaturated side chain at position 1 and ortho-substituted methoxy moiety on 4-phenoxyethylamine.     

Electron-paramagnetic-resonance spectroscopy of complexes of xanthine oxidase with xanthine and uric acid.

Rat fibrinogen was purified from rat plasma by using lysine–Sepharose chromatography, repeated precipitation with 25%-satd. (NH₄)₂SO₄ and gel chromatography on Sepharose 6B. To minimize proteolytic activity, rats were injected intravenously with Trasylol before bleeding and the collected blood was treated with Trasylol and di-isopropyl phosphorofluoridate. A 95%-clottable preparation was obtained in 70–75% yield; it proved to be free of factor XIII and plasminogen. It showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and on disc electrophoresis in 8m-urea. Alanine was the only detectable N-terminal amino acid. After reduction and modification of the thiol groups, the material could be separated into three distinct chains (Aα, Bβ and γ) by pore-limit polyacrylamide slab-gel electrophoresis in the presence of sodium dodecyl sulphate. The amino acid compositions of the whole fibrinogen and of the separated modified chains were determined. The molecular weights were 61000, 58000 and 51000 for Aα-, Bβ- and γ-chains respectively. Our results for the chains are in contrast with previous reports on rat fibrinogen [Bouma & Fuller (1975) J. Biol. Chem. 250, 4678–4683; Stemberger & Jilek (1976) Thromb. Res. 9, 657–660], in which no separation between Aα- and Bβ-chains was achieved on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis for 3h. Evidence is presented that this is probably due to Aα-chain degradation as a result of incomplete inhibition of proteolytic enzymes during the purification. Complete inhibition of proteolytic activities is essential in all steps of the present purification procedure.     

Phytic acid. A natural antioxidant

The catalysis by iron of radical formation and subsequent oxidative damage has been well documented. Although many iron-chelating agents potentiate reactive oxygen formation and lipid peroxidation, phytic acid (abundant in edible legumes, cereals, and seeds) forms an iron chelate which greatly accelerates Fe2+-mediated oxygen reduction yet blocks iron-driven hydroxyl radical generation and suppresses lipid peroxidation. Furthermore, high concentrations of phytic acid prevent browning and putrefaction of various fruits and vegetables by inhibiting polyphenol oxidase. These observations indicate an important antioxidant function for phytate in seeds during dormancy and suggest that phytate may be a substitute for presently employed preservatives, many of which pose potential health hazards.     

A new aspect of the RNA bacteriophages translation control mechanism.

The polarity effect of the coat protein gene of the ribonucleic acid of RNA bacteriophages on the polymerase gene translation will be taken as the basis of the polymerase translation control mechanism. A further condition for this mechanism discussed in this work is the dependence of the phage RNA replication on host cell translation factors. The ribosome binding sites of the phage RNA play a decisive role to realize the control mechanism coding for definite ribosome binding probabilities. The relation between them quantifies the reached polymerase concentration in the early phase of the development of the RNA bacteriophage system in the infected cell.     

Action of biologically-relevant oxidizing species upon uric acid. Identification of uric acid oxidation products

Uric acid is an end-product of purine metabolism in Man, and has been suggested to act as an antioxidant in vivo. Products of attack upon uric acid by various oxidants were measured by high performance liquid chromatography. Hypochlorous acid rapidly oxidized uric acid, forming allantoin, oxonic/oxaluric and parabanic acids, as well as several unidentified products. HOCl could oxidize all these products further. Hydrogen peroxide did not oxidize uric acid at detectable rates, although it rapidly oxidized oxonic acid and slowly oxidized allantoin and parabanic acids. Hydroxyl radicals generated by hypoxanthine/xanthine oxidase or Fe2(+)-EDTA/H2O2 systems also oxidized uric acid to allantoin, oxonic/oxaluric acid and traces of parabanic acid. Addition of ascorbic acid to the Fe2(+)-EDTA/H2O2 system did not increase formation of oxidation products from uric acid, possibly because ascorbic acid can 'repair' the radicals resulting from initial attack of hydroxyl radicals upon uric acid. Mixtures of methaemoglobin or metmyoglobin and H2O2 also oxidized uric acid: allantoin was the major product, but some parabanic and oxonic/oxaluric acids were also produced. Caeruloplasmin did not oxidize uric acid under physiological conditions, although simple copper (Cu2+) ions could, but this was prevented by albumin or histidine. The possibility of using oxidation products of uric acid, such as allantoin, as an index of oxidant generation in vivo in humans is discussed.     

Identification of a new candidate locus for uric acid nephrolithiasis

Renal stone formation is a common multifactorial disorder, of unknown etiology, with an established genetic contribution. Lifetime risk for nephrolithiasis is approximately 10% in Western populations, and uric acid stones account for 5%-10% of all stones, depending on climatic, dietary, and ethnic differences. We studied a small, isolated founder population in Sardinia, characterized by an increased prevalence of uric acid stones, and performed a genomewide search in a deep-rooted pedigree comprising many members who formed uric acid renal stones. The pedigree was created by tracing common ancestors of affected individuals through a genealogical database based on archival records kept by the parish church since 1640. This genealogical information was used as the basis for the study strategy, involving screening for alleles shared among affected individuals, originating from common ancestors, and utilization of large pedigrees to obtain greater power for linkage detection. We performed multistep linkage and allele-sharing analyses. In the initial stage, 382 markers were typed in 14 closely related affected subjects; interesting regions were subsequently investigated in the whole sample. We identified two chromosomal regions that may harbor loci with susceptibility genes for uric acid stones. The strongest evidence was observed on 10q21-q22, where a LOD score of 3.07 was obtained for D10S1652 under an affected-only dominant model, and a LOD score of 3.90 was obtained using a dominant pseudomarker assignment. The localization was supported also by multipoint allele-sharing statistics and by haplotype analysis of familial cases and of unrelated affected subjects collected from the isolate. In the second region on 20q13.1-13.3, multipoint nonparametric scores yielded suggestive evidence in a approximately 20-cM region, and further analysis is needed to confirm and fine-map this putative locus. Replication studies are required to investigate the involvement of these regions in the genetic contribution to uric acid stone formation.     

Electron paramagnetic resonance of copper ion and manganese ion complexes with the ionophore A23187.

The binding of Cu2+ and Mn2+ to the ionophore A23187 in chloroform, 90% ethanol, and sonicated phospholipid dispersions in aqueous mediums has been investigated with electron paramagnetic resonance (epr). The spectra indicated axial symmetry for the Cu2+ complexes and distorted octahedral for the Mn2+ complexes. The coordination between metal ion and its ligands is predominantly ionic in character. The stoichiometry, at the concentrations employed, was found to be 1:2 M2+/ionophore except in 90% ethanol where evidence existed for the 1:1 Cu-A23187 complex, as well. Through competition with Mn2+, the sequence of relative affinities in 90% ethanol was measured to be: Mn2+ greater than La3+ greater than Cu2+ greater than Ca2+ greater than Mg2+ greater than Sr2+. The K A of Mn-A23187 binding is greater than 10 10 M-2. In phospholipid dispersions the spectral characteristics of the Cu complex, particularly g, were observed to be a sensitive function of the hydrocarbon chain mobility. This allowed a calculation of the rotational correlation time of the complex to be made. In sonicated dipalmitoyllecithin was computed to be 10-9 sec, reflecting a local viscosity similar to that sensed by the nitroxide spin-label 2,2,6,6-tetramethylpiperidin-1-oxyl. In a (1:1) lecithin-cholesterol dispersion the complex was significantly more immobilized.     

Metal ion complexes of cytidinediphosphocholine.

Cytidine-5′-diphosphocholine (CDPcholine) forms a complex with Mg²⁺ and Mn²⁺ ions as indicated by the broadened ³¹P NMR peaks of CDPcholine in the presence of these ions. Additional evidence for the complex is the decrease in absorption at 360 nm when CDPcholine is added to solutions of 8-hydroxyquinoline and Mg²⁺. The stability constant of the Mg-CDPcholine complex was found to be 20 M^?1.     

Adverse effects of the classic antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/nitrosative stress

Uric acid is considered a major antioxidant in human blood that may protect against aging and oxidative stress. Despite its proposed protective properties, elevated levels of uric acid are commonly associated with increased risk for cardiovascular disease and mortality. Furthermore, recent experimental studies suggest that uric acid may have a causal role in hypertension and metabolic syndrome. All these conditions are thought to be mediated by oxidative stress. In this study we demonstrate that differentiation of cultured mouse adipocytes is associated with increased production of reactive oxygen species (ROS) and uptake of uric acid. Soluble uric acid stimulated an increase in NADPH oxidase activity and ROS production in mature adipocytes but not in preadipocytes. The stimulation of NADPH oxidase-dependent ROS by uric acid resulted in activation of MAP kinases p38 and ERK1/2, a decrease in nitric oxide bioavailability, and an increase in protein nitrosylation and lipid oxidation. Collectively, our results suggest that hyperuricemia induces redox-dependent signaling and oxidative stress in adipocytes. Since oxidative stress in the adipose tissue has recently been recognized as a major cause of insulin resistance and cardiovascular disease, hyperuricemia-induced alterations in oxidative homeostasis in the adipose tissue might play an important role in these derangements.     

Insulin neuroprotection against oxidative stress in cortical neurons--involvement of uric acid and glutathione antioxidant defenses

In this study we investigated the effect of insulin on neuronal viability and antioxidant defense mechanisms upon ascorbate/Fe2+-induced oxidative stress, using cultured cortical neurons. Insulin (0.1 and 10 microM) prevented the decrease in neuronal viability mediated by oxidative stress, decreasing both necrotic and apoptotic cell death. Moreover, insulin inhibited ascorbate/Fe2+-mediated lipid and protein oxidation, thus decreasing neuronal oxidative stress. Increased 4-hydroxynonenal (4-HNE) adducts on GLUT3 glucose transporters upon exposure to ascorbate/Fe2+ were also prevented by insulin, suggesting that this peptide can interfere with glucose metabolism. We further analyzed the influence of insulin on antioxidant defense mechanisms in the cortical neurons. Oxidative stress-induced decreases in intracellular uric acid and GSH/GSSG levels were largely prevented upon treatment with insulin. Inhibition of phosphatidylinositol-3-kinase (PI-3K) or mitogen-induced extracellular kinase (MEK) reversed the effect of insulin on uric acid and GSH/GSSG, suggesting the activation of insulin-mediated signaling pathways. Moreover, insulin stimulated glutathione reductase (GRed) and inhibited glutathione peroxidase (GPx) activities under oxidative stress conditions, further supporting that insulin neuroprotection was related to the modulation of the glutathione redox cycle. Thus, insulin may be useful in preventing oxidative stress-mediated injury that occurs in several neurodegenerative disorders.     

Reaction of uric acid with peroxynitrite and implications for the mechanism of neuroprotection by uric acid

Peroxynitrite, a biological oxidant formed from the reaction of nitric oxide with the superoxide radical, is associated with many pathologies, including neurodegenerative diseases, such as multiple sclerosis (MS). Gout (hyperuricemic) and MS are almost mutually exclusive, and uric acid has therapeutic effects in mice with experimental allergic encephalomyelitis, an animal disease that models MS. This evidence suggests that uric acid may scavenge peroxynitrite and/or peroxynitrite-derived reactive species. Therefore, we studied the kinetics of the reactions of peroxynitrite with uric acid from pH 6.9 to 8.0. The data indicate that peroxynitrous acid (HOONO) reacts with the uric acid monoanion with k = 155 M(-1) s(-1) (T = 37 degrees C, pH 7.4) giving a pseudo-first-order rate constant in blood plasma k(U(rate))(/plasma) = 0.05 s(-1) (T = 37 degrees C, pH 7.4; assuming [uric acid](plasma) = 0.3 mM). Among the biological molecules in human plasma whose rates of reaction with peroxynitrite have been reported, CO(2) is one of the fastest with a pseudo-first-order rate constant k(CO(2))(/plasma) = 46 s(-1) (T = 37 degrees C, pH 7.4; assuming [CO(2)](plasma) = 1 mM). Thus peroxynitrite reacts with CO(2) in human blood plasma nearly 920 times faster than with uric acid. Therefore, uric acid does not directly scavenge peroxynitrite because uric acid can not compete for peroxynitrite with CO(2). The therapeutic effects of uric acid may be related to the scavenging of the radicals CO(*-)(3) and NO(*)(2) that are formed from the reaction of peroxynitrite with CO(2). We suggest that trapping secondary radicals that result from the fast reaction of peroxynitrite with CO(2) may represent a new and viable approach for ameliorating the adverse effects associated with peroxynitrite in many diseases.     

A new aspect of the protective functions of the blood-brain barrier: activities of four drug-metabolizing enzymes in isolated rat brain microvessels

The multiple functions of the blood-brain barrier (BBB) consist mostly in membrane properties controlling the bidirectional exchange of molecules between the general circulation and the central nervous system. As lipophilic molecules are able to easily penetrate the membrane of brain microvessels endothelial cells, an Achilles' heel exists in the brain's protective system. We measured the activities of some enzymes involved in the metabolism of lipophilic xenobiotics, i.e. cytochrome P-450-linked monooxygenases, epoxide hydrolase, NADPH:cytochrome P-450 reductase and 1-naphthol UDP-glucuronosyl transferase in isolated rat brain microvessels. The relatively high activities observed indicate the capacity of endothelial cells to metabolize xenobiotics, and, thus, to give an additional protection to the brain.